University Hospital ofJacksonville, Jacksonville, Florida 322092; Food and Drug Administration, .... many phage-typing programs around the world have become.
Vol. 30, No. 1
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1992, p. 252-254
0095-1137/92/010252-03$02.00/0 Copyright C) 1992, American Society for Microbiology
Phage Pattern-Specific Oxacillin-Resistant and Borderline Oxacillin-Resistant Staphylococcus aureus in U.S. Hospitals: Epidemiological Significance CHARLES H. ZIERDT,l* IAN K. HOSEIN,2 ROXANNE SHIVELY,3 AND JAMES D. MACLOWRY4 Microbiology Service, Clinical Pathology Department, National Institutes of Health, Bethesda, Maryland 208921; University Hospital of Jacksonville, Jacksonville, Florida 322092; Food and Drug Administration, Rockville, Maryland 208503; and Group Health Association, Inc., Washington, D.C. 200064 Received 22 April 1991/Accepted 30 September 1991
For a 13-year period (1978 through 1990), oxacillin-resistant (MIC, >4 ,ug/ml) Staphylococcus aureus (ORSA) strains were collected from Clinical Center (National Institutes of Health) patients and patients from five other U.S. hospitals. From Clinical Center patients, 251 of 253 isolates (99%) were bacteriophage typed as phage group III. Five other hospitals contributed 203 ORSA strains, of which 188 (93%) were group III. The group III ORSA strains predominantly included a characteristic core pattern of phages, 7147153/54/75/77. For the low-level (borderline) oxacillin-resistant strains (MIC, 2 to 4 ,ug/ml), amoxicillin-clavulanic acid combination (Augmentin) testing disclosed 62 hyper-4-lactamase producers, of which 59 (95%) were of a separate, distinct S. aureus strain, with the phage pattern 92/94/96/292/D-11 (group V). Thus, ORSA and hyper-j(lactamase producing S. aureus are distinct epidemic strains. were received from Veterans Administration (VA) Hospital, Sioux Falls, S. Dak., courtesy of Mary Jo Jaqua; 5 isolates were received from Walter Reed Army Medical Center, courtesy of Alan Cross; 7 isolates were received from VA Hospital, Loma Linda, Calif., courtesy of Harry Elston; and 44 isolates were received from the University of Chicago Hospital, courtesy of Mary Fran Smaron. Clinical Center, National Institutes of Health, contributed 253 isolates. HBLPSA. Five isolates were received from the Centers for Disease Control, courtesy of Robert C. Cooksey. All ORSA or HBLPSA isolates submitted were retested in the National Institutes of Health laboratory to confirm the category of oxacillin resistance. Sixty-two HBLPSA isolates (one isolate per separate patient per year) were recovered in the Clinical Center in 4 years (1987 through 1990). All isolates were tested with oxacillin, and so they will be referred to as ORSA in this study. A standardized suspension of morphologically similar colonies was inoculated into calcium- and magnesium-supplemented Mueller-Hinton broth to achieve a final concentration of 5 x 105 CFU/ml. The test procedure recommended by the manufacturer was followed in preparing Sensititre custom air-dried microtiter antibiotic-susceptibility test panels (Sensititre Microbiology Systems, Radiometer America Inc., Westlake, Ohio), which included oxacillin plus 2% NaCl and amoxicillin-clavulanic acid. The agar diffusion test used a 1-,ug oxacillin disk. Test panels were incubated at 35°C in 5% CO2 for 18 to 24 h. Results were read visually. In addition, all of the ORSA isolates were resistant by MIC determination to a concentration of 32 ,ug of amoxicillin and 16 ,ug of clavulanic acid per ml, and the HBLPSA isolates were susceptible at a concentration of 4 ,ug of amoxicillin and 2 jig of clavulanic acid per ml. Phage typing. A total of 29 bacteriophages at 100 routine test doses were used: 3B, 3C, 6, 7, 29, 42B, 42E, 44A, 47, 52, 52A, 53, 54, 55, 71, 75, 77, 80, 81, 83, 86, 92, 94, 95, 96, 99, 187, 292, and D-11. Group III phages within the set included 6, 7, 42B, 42E, 47, 53, 54, 75, 77, and 83. Group V phages included 92, 94, 96, 292, and D-11. A lysis pattern of one or
The first Staphylococcus aureus strains shown resistant to methicillin were not from methicillin-treated patients. These strains possessed natural resistance to this drug (1, 2, 10, 14). Of 5,440 S. aureus strains collected from England before methicillin usage, 3 were methicillin resistant (10). All three had a group III phage pattern, 7/47/53154175177. Barber reported in 1964 (2) on natural methicillin-resistant S. aureus among strains collected from hospitals in England, France, and Denmark. Knox (14) and Sutherland and Rolinson (22) described the characteristics of methicillin-resistant S. aureus. These also had the same general phage pattern, 7/47/ 53/54/75/77. Pattern length variation for a given group III strain was and is common (18). About 1968, a marked increase of clinically significant methicillin-resistant S. aureus isolates was noted in England (19). Methicillin-resistant S. aureus infections in the United States increased dramatically in the late 1970s and reached epidemic proportions in the early 1980s. This increase was noted in four large hospital centers, affiliated with medical schools (8). Boston City Hospital had no methicillin-resistant strains until 1967 (3), but in the succeeding 12 months, 22 strains were isolated from 18 patients. The present study uncovered additional evidence that the group III strain(s) described in the first 1960s reports continues into the 1990s as a nosocomial pathogen. Oxacillin resistant S. aureus (ORSA) strains characteristically possess a distinct orange-yellow pigmentation, have much higher catalase production than non-ORSA strains, and have increased resistance to Tween 80 and mercuric chloride (20, 22). Borderline oxacillin-resistant S. aureus strains (6, 16) have been shown in this study to be hyper-fi-lactamase-producing S. aureus (HBLPSA) strains (17) whose resistance is greatly neutralized in the presence of clavulanic acid. HBLPSA strains from a number of geographical areas in the United States were phage typed. ORSA. From outside sources, 39 isolates were received from University Hospital of Jacksonville, Fla.; 108 isolates *
Corresponding author. 252
NOTES
VOL. 30, 1992
TABLE 1. ORSA in the Clinical Centeir, by year
'
No. of ORSA isolates
ai
V.r-ar Y
GGroup HPl
Nottyped phage
2 22
1
1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990
1 14 2 18 25 30 12 10 30 46 50 4 9
1 12 8 27 21 19 28 19 25 1 0
Total
251
171
___ Phage typed at one/patient/yr 1 2 1 6 9 8
Total
9
3 30 33
57 33 29 58
5 8
18
65 75 5 9
12 3 7
422
82
patient was infected with two ORSA strains in groupsth This patient had one ORSA strain with the phage pattern 3B/3C/ 55/71 and one ORSA strain with the phage pattern 29/52, /80. Therefore, of the 84 phage-typed ORSA isolates (one/patient/year). 82 (9M 8%) were gr-oup III. a
One
group
Ill.
phages of a single group places the tested S. aureus strain within that group. Given the large nunnber of group III phages (i.e., 10), a firm strain differentiation within the group phages requires a matching pattern, usu ally within two phages in length, and core or common phatges ot the same numbers. Core phages for the studied strain iwere 7/47/53/54/ 75/77. To minimize the risk of phage mutation dutring propagation and to ensure the same concentration of v,iral particles for succeeding typing runs, phage lots were e lyophilized in aliquots (25). After postlyophilization titer ing, the phages, sealed in glass vials under vacuum, were stc red at --200C, at which the titers remained stable for at lea st 18 yeais (23). Sufficient vials of each phage were stored to supply 15 years of phage typing, performed once month.-f 'ryptic soy broth used througho ut this study for of the same lot number growing the bacterial strains before tylping. For agar, SeaKemn agarose of the same lot number wt as used throughout, conmbined with tryptic soy broth at 1V /c wt/vol. Anhydrous calcium chloride (147 mg/liter) was; added. Eagle's vitamin mix (100 x) was added at 1%. 2 A bacteriophage applicator (24) was used. Other facets of the technique were previously published (25). Square petri plzates (100 by 100 mm) were used, each plate receiving 40 ml of imiolten agar. Clinical Center ORSA strains (Table 1) ha.d predomninantly a group III lytic pattern. One patient carrie :d three different phage-patterned ORSA strains, one of whicch, the group III straiti, was recovered from blood over a 2-- ieek period. One ORSA was of group I and one was of gloup II. 'Ihe possibility of resistance transfer between thiis patient's three separate ORSA strains exists. Many pati ents carried the group III epideniic ORSA for a numnber ot years anid were infected repeatedly with their endogenous ist4ain. Atnong Clinical Center HBLPSA strain s, 59 of the 62 (95%) borderline oxacillin-resistatit str;ains (HBLPSA straiins) had the group V phage pattern 922U94/96/292/D-11. The remaining three strains had phage patte rns 42E/44A/47, 47, and 3B/3C/55. (Of the 62 strains, 6 were c;ollected in 1987, 16 were collected in 1988, 24 were collectecI in 1989, and 16 were collected in 1990.) For four isolates, tIhe oxacillin MIC was >4 ,ug/ml, but these isolates were susc eptible - - to - - atnoxx-more
a
was
-
---
253
acillin-clavulanic acid. Their phage pattern, 92194/9612921D11, also indicated that these strains were HBLPSA, and they were included in that group. Of the five HBLPSA isolates from the Centers for Disease Control, three were group V, and two were nontypeable. Of 89 Clinical Center patients, 7 carried both the ORSA and the HBLPSA strains. Of the 253 ORSA strains (National Institutes of Health), 251 (99%) were group III. The University Hospital, Jacksonville, strains were all (39 of 39) group III. 'rhe VA Hospital, Sioux Falls, ORSA strains were 94% group III (102 of 108), with six strains (5%) being nontypeable. For the ORSA strains from University of Chicago Hospital, 36 of 44 (82%) were group IlI, with 8 (18%) being nontypeable. VA Hospital, Loma Linda, had six of seven strains (87%) that were group IlI, with one (14%) that was nontypeable, and five of five (100%) ORSA strains from Walter Reed Hospital were group III. Combined ORSA strains from the six hospitals were 96% group III (439 of 456). Although bacteriophage typing of S. aureus strains is the most sensitive tool available for epidemniological study, many phage-typing programs around the world have become less useful because of viral mutation during continuous phage propagations. Reactions from phage sets at separate laboratories vary so much, particularly for the older group III phages, that it is difficult to evaluate data from different institutions. In this laboratory, phage lyophilized in aliquots and stored in vacuo at -- 20°C permitted use of the same phage propagation lot for about 15 years of monthly typing (23). Most propagations in ouI laboratory require only one passage. In 1961, Barber (1) reported phage patterns for the first three methicillin-resistant S. aureus strainis found in England. The patterns were 53/54/75/77, 7/47/53/75/77, and 7/47/53/54/75/77. The ORSA strains reported here have the same general patterns as those reported by Barber. It is reasonable to assume that present-day ORSA strains are derived trom the worldwide spread of closely related epidemic ORSA strains. '[his is supported by numerous reports from around the world of similar group IlI-patterned ORSA strains (3-9, 11, 13, 15, 16, 18-20). Other strains have arisen, some with gr-oup I phage patterns (20), represented by phage pattern 29/52/80. Group II tiethicillin-resistant S. aureus phage patterns are seen less often and are represented by selections from phages 3A/3B/ 3C/55/71 (12). During a detailed 1981 study of three epidemic ORSA strains at the University of Virginia, Peacock et al. (20) recorded the phage pattern 6/47/54/75/83A and a MIC of >128 ,ug/ml (methicillin) for one strain. The MIC for the second strain was 2.0 p.g/ml, and the phage pattern was 96; the MIC for the third strain was 2.0 p.g/ml and the phage pattern was 94/96. The latter two strains were probably HBLPSA stiains. This established that yet another U.S. hospital has had the pandemic, specific-phage-patterned ORSA and HBLPSA strains introduced. Fromi the evidence presented in this paper, it seerns likely that these two distinct strains represent the predominant ORSA strain in many U.S. hospitals and the predominant HBLPSA strain (92/94/96/292/ D-11). Most phage typing sets do not include all phages of this lytic group of phages. Schaefler et al. (21) used only phage 92. However, susceptibility to even one phage of the group places the S. aureus strain within this closely related group of phages. IThus, the strains reported as methicillinresistant phage 92 strains by Schaefler may have been borderline resistant or HBLPSA strains. The level of resis-
254
NOTES
tance was not given. If so, since Schaefler studied strains from a number of New York City hospitals, the HBLPSA strain is also widely disseminated in the New York City area. It is apparent that the group III and group V phage patterns constitute positive markers for the predominant worldwide ORSA strains and in the United States, at least, for HBLPSA strains. ORSA strains were present in very small numbers before the advent of methicillin or oxacillin. These antibiotics paved the way for an increase in ORSA strains by eliminating susceptible S. aureus. The general increase and subsequent decrease in the incidence of ORSA infections in most areas of the world, with notable exceptions in individual hospitals and localized areas, follows the epidemiologic characteristics of previous S. aureus pandemics. Among these are the postantibiotic sloughing enteritis of the 1950s attributed to phage group III S. aureus and the devastating pandemic of generalized infections in the 1950s and early 1960s caused by the phage group I S. aureus, phage pattern 80/81 (9). Transmission of the S. aureus phage core pattern 7/47/53/54/75/77 around the world is similar to that of S. aureus phage pattern 80/81, which originated in 1954 in Australia. S. aureus 80/81, of course, was penicillin resistant and left an unparalleled path of serious infections, with mortality exceeding that of any strain on record. Perhaps if the few ORSA strains had not been present at the advent of methicillin and oxacillin therapy, no significant resistance to these drugs would have been encountered over the years.
J. CLIN. MICROBIOL.
9. 10. 11.
12.
13.
14.
15.
16.
17. For their contributions to the manuscript, we thank Barbara Fahey of our Epidemiology Service, Alice Faust of our Computer Service, and Gary Fahle and Dene Zierdt of our Microbiology Service.
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